Luteinizing hormone (LH) and follicle stimulating hormone (FSH) are released from the anterior pituitary gland under the control of luteinizing releasing hormone (LHRH) produced in the hypothalamic region. LH and FSH act on the gonads to stimulate the synthesis of steroid hormones and to stimulate gamete maturation. The pulsatile release of LHRH, and thereby the release of LH and FSH, controls the reproductive cycle in domestic animals and humans.
LHRH also affects the placenta, and therefore the gonads indirectly, by causing the synthesis and release of chorionic gonadotropin (CG).
Antagonists of LHRH are useful for the control of fertility. Such antagonists block ovulation in the female and suppress spermatogenesis in the male. Related to these effects is a suppression of normal circulating levels of sexual steroids of gonadal origin, causing reduction in accessory organ weight in the male and the female. In domestic animals this effect suppresses sexual cycling and behavior (promoting weight gain in a feed-lot situation), induces abortion in pregnant animals, and in general, acts as a chemical sterilant.
The natural releasing hormone LHRH is a decapeptide comprised of naturally occuring amino acids (which have the L-configuration except for the achiral amino acid glycine). Its sequence is as follows: ##STR1## Analogs of this natural material are often described in abbreviated form by showing the nature of the substituent of a given amino acid, superscribed by its location, followed by "LHRH." Many analogs of LHRH have been studied, and a very large majority are of insufficient biological activity to be clinically useful. However, certain select modifications have a potentiating effect on agonist biological activity. A significant enhancement to agonist activity is obtained by changing the 6-position residue from Gly to a D-amino acid.
In addition to agonists, analogs have been prepared which are competitive antagonists to LHRH, all of which require deletion or replacement of the histidyl residue at position 2: Vale, W., et al., Science, 176: 933 (1972). In general, it appears that a D-amino acid placed in the sequence at that position gives the best activity: Rees, R. W. A., et al., J. Med. Chem. 17: 1016 (1974).
Moreover, adding a modification at the 6 position (which, without the modification at position 2, results in the agonist activity cited above) enhances the antagonist activity of the 2-modified analogs: Beattie, C. W., et al, J. Med. Chem., 18: 1247 (1975); Rivier, J., et al., Peptides 1976 p. 427, Editions de l'Universite de Bruxelles, Belgium (1976).
Against the background of these two major alterations, which result in more potent LHRH antagonists, additional increments in antagonist activity may be had by modifying positions 1, 3, 5 and/or 10 in the already 2, 6 modified peptide. Coy, D. H., et al Peptides 1976, p. 462, Editions de l'Universite de Bruxelles, Belgium (1976); Rivier, J. E., et al, Life Sci. 23: 869 (1978); Dutta, A. S., et al, Biochem Biophys. Res. Commun. 81: 382 (1978), Humphries, J., et al, Biochem. Biophys. Res. Commun., 85: 709 (1978). It has also been shown that N-acylation of the amino acid at position 1 is helpful; Channabasavaia, K., et al, Biochem. Biophys. Res. Commun. 81: 382 (1978); Coy, D. H., et al, Peptides.--Structure and Biological Function p. 775, Pierce Chemical Co. (1979). Additionally, a highly potent antagonist containing a D-Arg.sup.6 substitution, (N-Ac-D-pCl-Phe.sup.1, D-pCl-Phe.sup.2, D-Trp.sup.3, D-Arg.sup.6, D-Ala.sup.10 )LHRH, has been published by D. H. Coy, Endocrinology, 110, 1445 (1982).
Unfortunately, although the class of LHRH analogs containing a D-Arg.sup.6 substitution were found to be potent antiovulatory substances, they were also potent mast cell degranulating substances, Schmidt et al., Contraception, 29, 283 (1984), and caused edema in vivo. Thus, for example, (N-Ac-D-Nal(2).sup.1, D-pCl-Phe.sup.2, D-Trp.sup.3, D-Arg.sup.6)LHRH has an ED.sub.50 =0.2 .mu.g/ml for histamine release from rat mast cells in vitro. This side reaction is of clinical importance because of the potential life threatening nature of the ensuing anaphylactoid reaction.
It is well known in the art that molecules containing positive charge(s), especially multiple positive charges, in association with hydrophobicity are potent mast cell degranulators: Foreman and Jordan, Agents and Actions, 13, 105 (1983). An initial attempt to circumvent this problem in analogs containing two Arg residues (i.e., in positions 6 and 8) was to increase the space between the residues (e.g., (N-Ac-D-Nal(2).sup.1, D-pCl-Phe.sup.2, D-Trp.sup.3, Arg.sup.5, D-Tyr.sup.6, D-Ala.sup.10)LHRH, (ED.sub.50 =2 .mu.g/ml for histamine release, R. Roeske, personal communication). While this led to a decrease in the potency of the analog for degranulation of mast cells and release of histamine, the analog still had many fold greater anaphylactoid potency than LHRH, for which ED.sub.50 is 328 .mu.g/ml for histamine release.
R. Roeske (personal communication) has incorporated Lys(iPr) into positions 6 and 8 in conjunction with a D-pCl-Phe residue in position 2 with retention of high antiovulatory potency and decreased histamine release (e.g., (N-Ac-D-Nal(2).sup.1, pCl-Phe.sup.2, D-Trp.sup.3, D-Lys(iPr).sup.8, Lys(iPr).sup.6, D-Ala.sup.10)LHRH; ED.sub.50 =6.6 .mu.g/ml for histamine release). In one analog, hArg(Et.sub.2) was incorporated into position 8 with a similar degree of histamine release potency (i.e., (N-Ac-D-Nal(2).sup.1, D-.alpha.Me-pCl-Phe.sup.2, D-Pal(3).sup.3, D-Arg.sup.6, hArg(Et.sub.2).sup.8, D-Ala.sup.10)LHRH; ED.sub.50 =4.9 .mu.g/ml for histamine release). However, it can be seen that these analogs are still potent histamine releasing agents compared to LHRH.
Thus, the presently known set of analogs still has significant possibility for toxicity and other side effects.